Novel next-generation sequencing assay for monitoring multidrug resistant tuberculosis treatment in the setting of HIV infection Multidrug resistant tuberculosis (MDR-TB) is a worsening global public health crisis and critical barrier to achieving TB elimination during our lifetimes. Current treatment of MDR-TB requires long treatment courses of decades-old, toxic, and poorly efficacious second-line drugs. In the setting of HIV co-infection, in particular, treatment of MDR-TB is complicated by extraordinary pill burden, overlapping drug toxicities, poor drug absorption, and often results in high early mortality. The extent of second-line anti-TB drug resistance during treatment is a strong predictor of poor outcome, but such resistance is only detected by existing molecular tests when it is already well-established. Evidence from our preliminary studies and others? suggest that small resistant M. tuberculosis (M.tb) subpopulations may be common precursors to clinical resistance. Detection and monitoring of micro-heteroresistance (<5% of total M.tb population, beneath the threshold for commonly used TB molecular tests) could transform clinical management through individualized treatment regimens and prompt reassessment of ineffective treatments (i.e., sub-therapeutic drug levels or inadequate regimens). Tremendous financial and scientific resources are directed toward investigation of new drugs for MDR-TB, but efforts to optimize and shorten treatment are hindered significantly by a poor understanding of exposure-response relationships for each drug within multi-drug regimens and how to best identify those patients who will respond inadequately to treatment. Our goal in proposing this work is to comprehensively characterize the pharmacologic correlates of M.tb micro-heteroresistance, and to determine the extent to which detection of micro- heteroresistance improves clinicians? ability to predict those MDR-TB patients, with and without HIV co-infection, who are at especially high likelihood for poor outcome. In order to achieve our Aims, we will leverage the infrastructure established through two major MDR-TB clinical trials, an existing NIH/NIAID R01 award, and the coordinated efforts of a large international non-profit organization whose mission is to enable development and delivery of diagnostic tests for poverty-related diseases.